Under Aim 1 of this project we have conducted necessary preclinical work for the initiation of a clinical trial testing CD22-targeted CAR T cells as a therapy for relapsed or resistant pre-B ALL. CD22 is expressed on 95 % of pre-B cell ALL and there is extensive expertise in the Pediatric Oncology Branch in CD22-targeted therapy for ALL using immunotoxin conjugated antibodies. This trial is currently open and utilizes lentiviral-based viral vectors for genetic modification of patient-derived expanded T cells to be reinfused after lymphodepleting chemotherapy. The study design is a phase I cell dose escalation study with an expansion cohort where an approximately 30% complete remission rate will used to determine whether this construct will be developed further. First, a number of important findings resulted from the pre-clinical work that have substantially streamlined the process of generating CAR T cells. Second, this trial will be critical in establishing whether the success of CD19 CAR T cell therapy can be extended to other targets on ALL. Third, since CD19 negative relapse has been observed following CD19 CAR therapy, a second target will potentially increase the overall curative potential of CAR therapy for ALL. This trial is open, has treated 6 patients and is expected to be completed in 1.5 years. We are also performing necessary pre-clinical work for the incorporation of an alternative, semi-automated T cell expansion and transduction that will further streamline the production process. Under Aim 2 of this project we have developed a number of novel CAR constructs including a TSLPR-targeted CAR for which we hold a patent. This work has been published in Blood and we are preparing for a clinical trial. In addition, we have generated a CD19/CD22 bispecific CAR with excellent in vivo activity. This construct can prevent the emergence of target-loss variants of ALL in an in vitro system. Second, we are developing novel acute myelogenous leukemia CAR constructs that may reduce the on-target off-tumor toxicity associated with myeloid antigens such as Flt3.